Methods for preventing or treating insulin resistance

ABSTRACT

The invention provides methods of preventing or treating insulin resistance in a mammalian subject. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide having at least one net positive charge; a minimum of four amino acids; a maximum of about twenty amino acids; a relationship between the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) wherein 3p m  is the largest number that is less than or equal to r+1; and a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p t ) wherein 2a is the largest number that is less than or equal to p t +1, except that when a is 1, p t  may also be 1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/367,267, filed Feb. 6, 2009, which claims priority to U.S.Provisional Patent Application No. 61/026,882, filed Feb. 7, 2008, theentire contents of which are hereby incorporated by reference in itsentirety.

GOVERNMENT RIGHTS

This invention was made with United States Government support awardedunder NIH grant DK073488 and DK056112. The United States Government hascertain rights in this invention.

TECHNICAL FIELD

The present invention relates generally to the methods of preventing ortreating insulin resistance. In particular, the present inventionrelates to administering aromatic-cationic peptides in effective amountsto prevent or treat insulin resistance in mammalian skeletal muscletissues.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Obesity has become a worldwide epidemic, the consequences of whichrepresent a major challenge facing human health in the 21st century. Adecrease in the sensitivity of skeletal muscle to insulin is one of theearliest maladies associated with obesity, and its persistence is aprominent risk factor for type II diabetes and cardiovascular disease.The accumulation of lipid in skeletal muscle has long been associatedwith the development of insulin resistance, a maladaptive response thatis currently attributed to the generation and intracellular accumulationof proinflammatory lipid metabolites (e.g., fatty acyl-CoAs,diacylglycerols and/or ceramides) and associated activation ofstress-sensitive serine/threonine kinases that antagonize insulinsignaling. Skeletal muscle from obese individuals is also characterizedby profound reductions in mitochondrial function as evidenced bydecreased expression of metabolic genes, reduced respiratory capacity,and mitochondria that are smaller and less abundant, leading tospeculation that a decrease in the capacity to oxidize fat due toacquired or inherited mitochondrial insufficiency may be an underlyingcause of the lipid accumulation and insulin resistance that develops invarious metabolic states.

SUMMARY

The present invention relates generally to the treatment or preventionof insulin resistance in skeletal muscle tissues through administrationof therapeutically effective amounts of aromatic-cationic peptides tosubjects in need thereof. In particular embodiments, thearomatic-cationic peptides treat or prevent diet-induced insulinresistance by reducing the occurrence of skeletal muscle mitochondrialdysfunction and over-production of reactive oxygen species.

In one aspect, the invention provides a method of treating or preventinginsulin resistance and related complications in a mammalian subject,comprising administering to said mammalian subject a therapeuticallyeffective amount of an aromatic-cationic peptide. In some embodiments,the aromatic-cationic peptide is a peptide having:

at least one net positive charge;

a minimum of four amino acids;

a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1; and arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be 1. In particular embodiments, the mammalian subject isa human.

In one embodiment, 2p_(m) is the largest number that is less than orequal to r+1, and a may be equal to p_(t). The aromatic-cationic peptidemay be a water-soluble peptide having a minimum of two or a minimum ofthree positive charges.

In one embodiment, the peptide comprises one or more non-naturallyoccurring amino acids, for example, one or more D-amino acids. In someembodiments, the C-terminal carboxyl group of the amino acid at theC-terminus is amidated. In certain embodiments, the peptide has aminimum of four amino acids. The peptide may have a maximum of about 6,a maximum of about 9, or a maximum of about 12 amino acids.

In some embodiments, the peptide has opioid receptor agonist activity.In other embodiments, the peptide does not have opioid receptor agonistactivity.

In one embodiment, the peptide comprises a tyrosine or a2′,6′-dimethyltyrosine (Dmt) residue at the N-terminus. For example, thepeptide may have the formula Tyr-D-Arg-Phe-Lys-NH₂ (SS-01) or2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (SS-02). In another embodiment, the peptidecomprises a phenylalanine or a 2′,6′-dimethylphenylalanine residue atthe N-terminus. For example, the peptide may have the formulaPhe-D-Arg-Phe-Lys-NH₂ (SS-20) or 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In aparticular embodiment, the aromatic-cationic peptide has the formulaD-Arg-2′6′Dmt-Lys-Phe-NH₂ (SS-31).

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In some embodiments, the aromatic-cationic peptides of the invention areused to treat or prevent complications related to insulin resistance inmammalian subjects, which include, but are not limited to,hyperinsulinemia, type II diabetes, abnormal lipid metabolism, abnormalvascular endothelial function, retinopathy, coronary artery disease,cardiovascular disease, renal dysfunction, hypertension, fatty liver,neuropathy, and hyperuricemia. Specific examples of cardiovasculardisease potentially caused by long-term insulin resistance includemyocardial infarction, hemorrhagic or ischemic stroke (cerebralinfarction).

The aromatic-cationic peptides of the invention may be administered in avariety of ways. In some embodiments, the peptides may be administeredorally, topically, intranasally, intravenously, subcutaneously, ortransdermally (e.g., by iontophoresis).

In another aspect, the invention provides a method of preventing and/ortreating diabetes, obesity, hyperlipemia, arteriosclerosis,cerebrovascular disease, hypertension or heart disease comprisingadministering a therapeutically effective amount of aromatic-cationicpeptides to subjects in need thereof. In a particular embodiment, thearomatic-cationic peptide comprises D-Arg-2′6′Dmt-Lys-Phe-NH₂ (SS-31).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a series of graphs showing the impact of excess dietary fat onskeletal muscle mitochondrial H₂O₂ emission. FIG. 1A is a representativetrace comparing rates of mitochondrial H₂O₂ emission from permeabilizedskeletal muscle fibers prepared from rats fed standard chow, high fatchow for 3 days, or high fat chow 3 weeks. FIG. 1B is a graph showingthe mitochondrial oxidant emitting potential of permeabilized ratskeletal myofibers in control rats fed a normal diet and rats fed a highfat diet.

FIG. 2 is a chart showing mitochondrial H₂O₂ emission in the presence ofantimycin A in skeletal muscle fibers prepared from high fat fed ratscompared to control chow. Mitochondrial H₂O₂ emission in the presence ofantimycin A was measured in both groups with a titration of pyruvate andmalate.

FIG. 3 is a series of charts showing the effect of the SS-31 peptide ofthe invention on the oxidation state of muscle tissues. Subjects includerats fed a normal diet (Ctl), a high fat diet (HF), or a high fat dietwith daily SS-31 administration (HF+SS-31). FIGS. 3A and 3B are graphsshowing dose response curves of SS-31 antioxidant activity in vitro andin vivo, respectively. FIGS. 3C and 3D are graphs showing themitochondrial oxidant emitting potential of permeabilized rat skeletalmyofibers in control rats fed standard chow, a high fat diet, or a highfat diet with daily SS-31 administration and assayed forsuccinate-stimulated H₂O₂ emission (FIG. 3C) and palmitoyl-carnitinestimulated H₂O₂ emission (FIG. 3D). FIGS. 3E and 3F are charts showingrespiration rates in control rats fed standard chow, a high fat diet, ora high fat diet with daily SS-31 administration. Respiration wasmeasured with pyruvate and malate (FIG. 3E) or palmitoyl-carnitine andmalate (FIG. 3F) in both basal (PM₄, PCM₄) and maximal ADP-stimulated(PM₃, PCM₃) respiratory states. FIG. 3G is a chart showing totalglutathione in control rats fed standard chow, a high fat diet, or ahigh fat diet with daily SS-31 administration. FIG. 3H is a chartshowing the ratio of GSH/GSSG in control rats fed standard chow, a highfat diet, or a high fat diet with daily SS-31 administration. Analyseswere performed either before (−) or 1 h after (+) oral glucoseingestion. Data are representative of mean±S.E.M.; n=4-6, *P<0.05 vs.Ctl-Std chow, †P<0.05 vs. SS-31 treated.

FIG. 4 is a series of charts showing the effect of the SS-31 peptide ofthe invention on the insulin resistance in muscle tissues. FIG. 4A is agraph showing plasma glucose clearance rate in control rats fed standardchow, a high fat diet, or a high fat diet with daily SS-31administration. FIG. 4B is a graph showing fasting plasma insulin levelsin the same experimental subjects. FIG. 4C is a chart showing increasedhomeostatic model assessment (HOMA) and FIG. 4D is a chart showinggreater area under the curve (AUC) for both glucose and insulin incontrol rats fed standard chow, a high fat diet, or a high fat diet withdaily SS-31 administration. Data are representative of mean±S.E.M.;n=9-10, *P<0.05 vs. Ctl-Std chow, †P<0.05 vs. SS31 treated. FIG. 4E isan immunoblot of phosphorylated Akt and total Akt in rat skeletal musclehomogenates in response to glucose challenge. FIG. 4F is a chart showingthe quantification of these blots using densitometry. Data arerepresentative of mean±S.E.M.; n=4-5, ‡P<0.05 vs. non-glucose challengedanimals.

FIG. 5 is a series of graphs showing the oxidation state of muscletissues from lean and obese human subjects. FIGS. 5A and 5B are graphsshowing mitochondrial H₂O₂ emission from permeabilized fibers preparedfrom biopsies obtained from vastus lateralis of obese and lean humanmales. Both succinate-supported (FIG. 5A) and palmitoyl-carnitinesupported (FIG. 5B) H₂O₂ emission were measured. FIG. 5C is a graphshowing the ratio of H₂O₂ emitted/O₂ consumed in obese samples ascompared to lean. FIG. 5D is a graph showing respiration in the presenceof glutamate/malate (GM₄), ADP (GM₃), and palmitoyl-carnitine/malate ineither basal (PCM₄) or maximal (PCM₃) state. FIG. 5E is a graph showingtotal glutathione (GSH_(t)) in obese skeletal muscle compared to lean.FIG. 5F is a graph showing the ratio of GSH/GSSG in obese skeletalmuscle compared to lean. Data are representative of mean±S.E.M.; n=4-5,*P<0.05 vs. lean male for that respective experiment.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention.

In practicing the present invention, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively. Methods to detect and measurelevels of polypeptide gene expression products (i.e., gene translationlevel) are well-known in the art and include the use polypeptidedetection methods such as antibody detection and quantificationtechniques. (See also, Strachan & Read, Human Molecular Genetics, SecondEdition. (John Wiley and Sons, Inc., NY, 1999)).

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like. Generally, thenomenclature used herein and the laboratory procedures in cell culture,molecular genetics, organic chemistry, analytical chemistry and nucleicacid chemistry and hybridization described below are those well knownand commonly employed in the art.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “biological sample” means sample materialderived from or contacted by living cells. The term “biological sample”is intended to include tissues, cells and biological fluids isolatedfrom a subject, as well as tissues, cells and fluids present within asubject. Biological samples of the invention include, e.g., but are notlimited to, whole blood, plasma, semen, saliva, tears, urine, fecalmaterial, sweat, buccal, skin, cerebrospinal fluid, and hair. Biologicalsamples can also be obtained from biopsies of internal organs or fromcancers. Biological samples can be obtained from subjects for diagnosisor research or can be obtained from undiseased individuals, as controlsor for basic research.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “effective amount” or “pharmaceuticallyeffective amount” or “therapeutically effective amount” of acomposition, is a quantity sufficient to achieve a desired therapeuticand/or prophylactic effect, e.g., an amount which results in theprevention of, or a decrease in, the symptoms associated with insulinresistance. The amount of a composition of the invention administered tothe subject will depend on the type and severity of the disease and onthe characteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. It will also depend on the degree,severity and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions of the present invention can also be administered incombination with one or more additional therapeutic compounds. In themethods of the present invention, the aromatic-cationic peptides may beadministered to a subject having one or more signs of insulin resistancecaused by a disease or condition. Administration of an effective amountof the aromatic-cationic peptides may improve at least one sign orsymptom of insulin resistance in the subject, e.g., body weight, fastingglucose/insulin/free fatty acid, glucose tolerance (OGTT), in vitromuscle insulin sensitivity, markers of insulin signaling (e.g., Akt-P,IRS-P), mitochondrial function (e.g., respiration or H₂O₂ emission),markers of intracellular oxidative stress (e.g., lipid peroxidation,GSH/GSSG ratio or aconitase activity) and mitochondrial enzyme activity.For example, a “therapeutically effective amount” of thearomatic-cationic peptides is meant levels in which the physiologicaleffects of insulin resistance are, at a minimum, ameliorated.

An “isolated” or “purified” polypeptide or peptide is substantially freeof cellular material or other contaminating polypeptides from the cellor tissue source from which the agent is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.For example, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, the term “medical condition” includes, but is notlimited to, any condition or disease manifested as one or more physicaland/or psychological symptoms for which treatment and/or prevention isdesirable, and includes previously and newly identified diseases andother disorders. For example, a medical condition may be insulinresistance, hyperinsulinemia, type II diabetes, abnormal lipidmetabolism, abnormal vascular endothelial function, coronary arterydisease, cardiovascular disease, cerebrovascular disease, renaldysfunction, hypertension, fatty liver, neuropathy, and hyperuricemia.

As used herein, the terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. A subject is successfully“treated” for insulin resistance if, after receiving a therapeuticamount of the aromatic-cationic peptides according to the methods of thepresent invention, the subject shows observable and/or measurablereduction in or absence of one or more signs and symptoms of aparticular disease or condition. For example, for insulin resistance,treatment may include a reduction in the fasting blood glucose orinsulin levels, or the areas under the curve for glucose and insulin inresponse to oral glucose challenge. It is also to be appreciated thatthe various modes of treatment or prevention of medical conditions asdescribed are intended to mean “substantial”, which includes total butalso less than total treatment or prevention, and wherein somebiologically or medically relevant result is achieved.

The present inventors have discovered that, surprisingly,aromatic-cationic peptides can prevent or treat insulin resistance inmammalian tissues; in particular, insulin resistance in skeletal muscletissues. In some cases, the insulin resistance may be due to a high fatdiet or, more generally, over-nutrition. The peptides of the inventionare beneficial in treating diabetic, pre-diabetic or obese insulinresistant, non-diabetic patients. Without intending to limit theinvention to a particular mechanism of action, it is believed that lossof mitochondrial integrity and insulin sensitivity stem from a commonmetabolic disturbance, i.e., oxidative stress. Over-nutrition,particularly from high fat diets may increase mitochondrial reactiveoxygen species (ROS) emission and overall oxidative stress in skeletalmuscle, leading to both acute and chronic mitochondrial dysfunction andthe development of insulin resistance. The aromatic-cationic peptides ofthe present invention mitigate these effects, thereby improvingmitochondrial function in skeletal muscle tissues, thus improvinginsulin sensitivity. The invention also provides methods of usingpeptides of the invention to prevent or treat diabetes, pre-diabetes,related metabolic diseases, and complications arising therefrom.

The present inventors found that high fat diet/obesity-induced insulinresistance is related to mitochondrial bioenergetics. The implication isthat the oversupply of metabolic substrates causes the mitochondrialrespiratory system to become more reduced, generating an increase in ROSemission and shift in the overall redox environment to a more oxidizedstate that, if persistent, leads to development of insulin resistance.Linking mitochondrial bioenergetics to the etiology of insulinresistance has a number of clinical implications. It is known thatstandard care of insulin resistance (NIDDM) in humans often results inweight gain and, in selected individuals, increased variability of bloodsugar with resulting metabolic and clinical consequences. The examplesshown herein demonstrate that treatment of mitochondrial defect withmitochondrial-targeted antioxidant (e.g. an aromatic cationic peptide)provides a new and surprising approach to metabolic correction ofinsulin resistance without the growth and metabolic effects of increasedinsulin.

The present invention relates to the reduction of insulin resistance bycertain aromatic-cationic peptides. The aromatic-cationic peptides arewater-soluble and highly polar. Despite these properties, the peptidescan readily penetrate cell membranes. The aromatic-cationic peptidesuseful in the present invention include a minimum of three amino acids,and preferably include a minimum of four amino acids, covalently joinedby peptide bonds. The maximum number of amino acids present in thearomatic-cationic peptides of the present invention is about twentyamino acids covalently joined by peptide bonds. Preferably, the maximumnumber of amino acids is about twelve, more preferably about nine, andmost preferably about six. Optimally, the number of amino acids presentin the peptides is four.

The amino acids of the aromatic-cationic peptides useful in the presentinvention can be any amino acid. As used herein, the term “amino acid”is used to refer to any organic molecule that contains at least oneamino group and at least one carboxyl group. Preferably, at least oneamino group is at the α position relative to a carboxyl group. The aminoacids may be naturally occurring. Naturally occurring amino acidsinclude, for example, the twenty most common levorotatory (L) aminoacids normally found in mammalian proteins, i.e., alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan, (Trp), tyrosine (Tyr), and valine (Val). Other naturallyoccurring amino acids include, for example, amino acids that aresynthesized in metabolic processes not associated with proteinsynthesis. For example, the amino acids ornithine and citrulline aresynthesized in mammalian metabolism during the production of urea.Another example of a naturally occurring amino acid includehydroxyproline (Hyp).

The peptides useful in the present invention optionally contain one ormore non-naturally occurring amino acids. Optimally, the peptide has noamino acids that are naturally occurring. The non-naturally occurringamino acids may be levorotary (L-), dextrorotatory (D-), or mixturesthereof. Non-naturally occurring amino acids are those amino acids thattypically are not synthesized in normal metabolic processes in livingorganisms, and do not naturally occur in proteins. In addition, thenon-naturally occurring amino acids useful in the present inventionpreferably are also not recognized by common proteases. Thenon-naturally occurring amino acid can be present at any position in thepeptide. For example, the non-naturally occurring amino acid can be atthe N-terminus, the C-terminus, or at any position between theN-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide usefulin the methods of the present invention is the derivatization of acarboxyl group of an aspartic acid or a glutamic acid residue of thepeptide. One example of derivatization is amidation with ammonia or witha primary or secondary amine, e.g. methylamine, ethylamine,dimethylamine or diethylamine. Another example of derivatizationincludes esterification with, for example, methyl or ethyl alcohol.Another such modification includes derivatization of an amino group of alysine, arginine, or histidine residue. For example, such amino groupscan be acylated. Some suitable acyl groups include, for example, abenzoyl group or an alkanoyl group comprising any of the C₁-C₄ alkylgroups mentioned above, such as an acetyl or propionyl group.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD-non-naturally occurring amino acids. The D-amino acids do not normallyoccur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell. As used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides useful in themethods of the invention should have less than five, preferably lessthan four, more preferably less than three, and most preferably, lessthan two contiguous L-amino acids recognized by common proteases,irrespective of whether the amino acids are naturally or non-naturallyoccurring. Optimally, the peptide has only D-amino acids, and no L-aminoacids. If the peptide contains protease sensitive sequences of aminoacids, at least one of the amino acids is preferably anon-naturally-occurring D-amino acid, thereby conferring proteaseresistance. An example of a protease sensitive sequence includes two ormore contiguous basic amino acids that are readily cleaved by commonproteases, such as endopeptidases and trypsin. Examples of basic aminoacids include arginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid.

Typically, a peptide has a positively charged N-terminal amino group anda negatively charged C-terminal carboxyl group. The charges cancel eachother out at physiological pH. As an example of calculating net charge,the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively chargedamino acid (i.e., Glu) and four positively charged amino acids (i.e.,two Arg residues, one Lys, and one His). Therefore, the above peptidehas a net positive charge of three.

In one embodiment of the present invention, the aromatic-cationicpeptides have a relationship between the minimum number of net positivecharges at physiological pH (p_(m)) and the total number of amino acidresidues (r) wherein 3p_(m) is the largest number that is less than orequal to r+1. In this embodiment, the relationship between the minimumnumber of net positive charges (p_(m)) and the total number of aminoacid residues (r) is as follows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≦ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≦ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, preferably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides useful in the methods of the presentinvention should also have a relationship between the minimum number ofaromatic groups (a) and the total number of net positive charges atphysiological pH (p_(t)) wherein 3a is the largest number that is lessthan or equal to p_(t)+1, except that when p_(t) is 1, a may also be 1.In this embodiment, the relationship between the minimum number ofaromatic groups (a) and the total number of net positive charges (p_(t))is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≦ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (3a ≦ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are preferably amidated with, for example, ammonia to formthe C-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides of thepresent invention may also be amidated wherever they occur within thepeptide. The amidation at these internal positions may be with ammoniaor any of the primary or secondary amines described above.

In one embodiment, the aromatic-cationic peptide useful in the methodsof the present invention is a tripeptide having two net positive chargesand at least one aromatic amino acid. In a particular embodiment, thearomatic-cationic peptide useful in the methods of the present inventionis a tripeptide having two net positive charges and two aromatic aminoacids.

Aromatic-cationic peptides useful in the methods of the presentinvention include, but are not limited to, the following peptideexamples:

Lys-D-Arg-Tyr-NH₂ Phe-D-Arg-His D-Tyr-Trp-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Tyr-His-D-Gly-Met Phe-Arg-D-His-Asp Tyr-D-Arg-Phe-Lys-Glu-NH₂Met-Tyr-D-Lys-Phe-Arg D-His-Glu-Lys-Tyr-D-Phe-ArgLys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂ Phe-D-Arg-Lys-Trp-Tyr-D-Arg-HisGly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysLys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysAsp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheTyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-PhePhe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysGlu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-GlyD-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-PheHis-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-AspThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂

In one embodiment, the peptides useful in the methods of the presentinvention have mu-opioid receptor agonist activity (i.e., they activatethe mu-opioid receptor). Mu-opioid activity can be assessed byradioligand binding assay to cloned mu-opioid receptors or by bioassayssuing the guinea pig ileum (Schiller et al., Eur J Med Chem, 35:895-901,2000; Zhao et al., J Pharmacol Exp Ther 307:947-954, 2003). Activationof the mu-opioid receptor typically elicits an analgesic effect. Incertain instances, an aromatic-cationic peptide having mu-opioidreceptor agonist activity is preferred. For example, during short-termtreatment, such as in an acute disease or condition, it may bebeneficial to use an aromatic-cationic peptide that activates themu-opioid receptor. Such acute diseases and conditions are oftenassociated with moderate or severe pain. In these instances, theanalgesic effect of the aromatic-cationic peptide may be beneficial inthe treatment regimen of the human patient or other mammal. Anaromatic-cationic peptide which does not activate the mu-opioidreceptor, however, may also be used with or without an analgesic,according to clinical requirements.

Alternatively, in other instances, an aromatic-cationic peptide thatdoes not have mu-opioid receptor agonist activity is preferred. Forexample, during long-term treatment, such as in a chronic disease stateor condition, the use of an aromatic-cationic peptide that activates themu-opioid receptor may be contraindicated. In these instances thepotentially adverse or addictive effects of the aromatic-cationicpeptide may preclude the use of an aromatic-cationic peptide thatactivates the mu-opioid receptor in the treatment regimen of a humanpatient or other mammal. Potential adverse effects may include sedation,constipation and respiratory depression. In such instances anaromatic-cationic peptide that does not activate the mu-opioid receptormay be an appropriate treatment.

Peptides useful in the methods of the present invention which havemu-opioid receptor agonist activity are typically those peptides whichhave a tyrosine residue or a tyrosine derivative at the N-terminus(i.e., the first amino acid position). Preferred derivatives of tyrosineinclude 2′-methyltyrosine (Mmt); 2′,6′-dimethyltyrosine (2′6′Dmt);3′,5′-dimethyltyrosine (3′5′Dmt); N,2′,6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltryosine (Hmt).

In one embodiment, a peptide that has mu-opioid receptor agonistactivity has the formula Tyr-D-Arg-Phe-Lys-NH₂ (referred to herein as“SS-01”). SS-01 has a net positive charge of three, contributed by theamino acids tyrosine, arginine, and lysine and has two aromatic groupscontributed by the amino acids phenylalanine and tyrosine. The tyrosineof SS-01 can be a modified derivative of tyrosine such as in2′,6′-dimethyltyrosine to produce the compound having the formula2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (referred to herein as “SS-02”). SS-02 has amolecular weight of 640 and carries a net three positive charge atphysiological pH. SS-02 readily penetrates the plasma membrane ofseveral mammalian cell types in an energy-independent manner (Zhao etal., J. Pharmacol Exp Ther. 304: 425-432, 2003).

Peptides that do not have mu-opioid receptor agonist activity generallydo not have a tyrosine residue or a derivative of tyrosine at theN-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (Dmp), N,2′,6′-trimethylphenylalanine (Tmp),and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of a aromatic-cationic peptide that does not have mu-opioidreceptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂(referred to herein as “SS-20”). Alternatively, the N-terminalphenylalanine can be a derivative of phenylalanine such as2′,6′-dimethylphenylalanine (2′6′Dmp). SS-01 containing2′,6′-dimethylphenylalanine at amino acid position 1 has the formula2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In one embodiment, the amino acid sequenceof SS-02 is rearranged such that Dmt is not at the N-terminus. Anexample of such an aromatic-cationic peptide that does not havemu-opioid receptor agonist activity has the formulaD-Arg-2′6′Dmt-Lys-Phe-NH₂ (SS-31).

SS-01, SS-20, SS-31, and their derivatives can further includefunctional analogs. A peptide is considered a functional analog ofSS-01, SS-20, or SS-31 if the analog has the same function as SS-01,SS-20, or SS-31. The analog may, for example, be a substitution variantof SS-01, SS-20, or SS-31, wherein one or more amino acids aresubstituted by another amino acid.

Suitable substitution variants of SS-01, SS-20, or SS-31 includeconservative amino acid substitutions. Amino acids may be groupedaccording to their physicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys(C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(O);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His(H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

In some embodiments, one or more naturally occurring amino acids in thearomatic-cationic peptides are substituted with amino acid analogs.Examples of analogs useful in the practice of the present invention thatactivate mu-opioid receptors include, but are not limited to, thearomatic-cationic peptides shown in Table 5.

TABLE 5 Peptide Analogs with Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid Position 5 C-Terminal Position 1 Position 2Position 3 Position 4 (if present) Modification Tyr D-Arg Phe Lys NH₂Tyr D-Arg Phe Orn NH₂ Tyr D-Arg Phe Dab NII₂ Tyr D-Arg Phe Dap NH₂2′6′Dmt D-Arg Phe Lys NH₂ 2′6'Dmt D-Arg Phe Lys-NII(CII₂)²⁻ NH₂ NH-dns2′6′Dmt D-Arg Phe Lys-NH(CH₂)²⁻ NH₂ NH-atn 2′6′Dmt D-Arg Phe dnsLys NH₂2′6′Dmt D-Cit Phe Lys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg PheOrn NH₂ 2′6′Dmt D-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′DmtD-Arg Phe Ahp(2- NII₂ aminoheptanoic acid) Bio-2′6′Dmt D-Arg Phe Lys NH₂3′5′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg PheDab NH₂ 3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg TyrOrn NH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg TyrLys NH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′DmtD-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′DmtOrn NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′DmtD-Arg 3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe DapNH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂2′6′Dmt D-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys PheArg NH₂ 2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′DmtD-Lys Phe Dab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NII₂Tyr D-Lys Tyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ TyrD-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂2′6′Dmt D-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys2′6′Dmt Lys NH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt DabNH₂ 2′6′Dmt D-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′DmtD-Arg Phe atnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′DmtOrn NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-DapPhe Arg NII₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Orn PheArg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr ArgNII₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt ArgNH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NII₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂Tmt D-Arg Phe Lys NH₂ Tmt D-Arg Phe Om NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-LysPhe Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NII₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NH₂ Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NII₂ Hmt D-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N, 2′,6′-trimethyltyrosine Hmt =2′-hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid atnDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of analogs useful in the practice of the present invention thatdo not activate mu-opioid receptors include, but are not limited to, thearomatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogs Lacking Mu-Opioid Activity Amino Amino AminoAmino Amino Amino Amino Acid Acid Acid Acid Acid Acid Acid C-TerminalPosition 1 Position 2 Position 3 Position 4 Position 5 Position 6Position 7 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl

The amino acids of the peptides shown in Table 5 and 6 may be in eitherthe L- or the D-configuration.

Synthesis of the Peptides

The peptides useful in the methods of the present invention may besynthesized by any of the methods well known in the art. Suitablemethods for chemically synthesizing the protein include, for example,those described by Stuart and Young in Solid Phase Peptide Synthesis,Second Edition, Pierce Chemical Company (1984), and in Methods Enzymol.289, Academic Press, Inc, New York (1997).

Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides.

General.

The aromatic-cationic peptides of the present invention are useful toprevent or treat disease. Specifically, the invention provides for bothprophylactic and therapeutic methods of treating a subject at risk of(or susceptible to) a disorder or having a disorder associated withinsulin resistance. Insulin resistance is generally associated with typeII diabetes, coronary artery disease, renal dysfunction,atherosclerosis, obesity, hyperlipidemia, and essential hypertension.Insulin resistance is also associated with fatty liver, which canprogress to chronic inflammation (NASH; “nonalcoholic steatohepatitis”),fibrosis, and cirrhosis. Cumulatively, insulin resistance syndromes,including, but not limited to diabetes, underlie many of the majorcauses of morbidity and death of people over age 40. Accordingly, thepresent invention provides methods for the prevention and/or treatmentof insulin resistance and associated syndromes in a subject comprisingadministering an effective amount of an aromatic-cationic peptide to asubject in need thereof. For example, a subject can be administered anaromatic-cationic peptide compositions of the present invention in aneffort to improve the sensitivity of mammalian skeletal muscle tissuesto insulin. In one embodiment, the aromatic-cationic peptides of theinvention are useful to prevent drug-induced obesity, insulinresistance, and/or diabetes, when the peptide is administered with adrug that shows a side-effect of causing one or more of these conditions(e.g., olanzapine, Zyprexa®).

Determination of the Biological Effect of the Aromatic-CationicPeptide-Based Therapeutic.

In various embodiments of the invention, suitable in vitro or in vivoassays are performed to determine the effect of a specificaromatic-cationic peptide-based therapeutic and whether itsadministration is indicated for treatment of the affected tissue in asubject. In various embodiments, in vitro assays can be performed withrepresentative cells of the type(s) involved in the subject's disorder,to determine if a given aromatic-cationic peptide-based therapeuticexerts the desired effect upon the cell type(s). Compounds for use intherapy can be tested in suitable animal model systems including, butnot limited to rats, mice, chicken, cows, monkeys, rabbits, and thelike, prior to testing in human subjects. Similarly, for in vivotesting, any of the animal model system known in the art can be usedprior to administration to human subjects. Increased or decreasedinsulin resistance or sensitivity can be readily detected by quantifyingbody weight, fasting glucose/insulin/free fatty acid, glucose tolerance(OGTT), in vitro muscle insulin sensitivity, markers of insulinsignaling (e.g., Akt-P, IRS-P), mitochondrial function (e.g.,respiration or H₂O₂ emission), markers of intracellular oxidative stress(e.g., lipid peroxidation, GSH/GSSG ratio or aconitase activity) ormitochondrial enzyme activity.

Prophylactic Methods.

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with insulin resistance inskeletal muscle tissues, by administering to the subject anaromatic-cationic peptide that modulates one or more signs or markers ofinsulin resistance, e.g., body weight, fasting glucose/insulin/freefatty acid, glucose tolerance (OGTT), in vitro muscle insulinsensitivity, markers of insulin signaling (e.g., Akt-P, IRS-P),mitochondrial function (e.g., respiration or H₂O₂ emission), markers ofintracellular oxidative stress (e.g., lipid peroxidation, GSH/GSSG ratioor aconitase activity) or mitochondrial enzyme activity.

Subjects at risk for a disease that is caused or contributed to byaberrant mitochondrial function or insulin resistance can be identifiedby, e.g., any or a combination of diagnostic or prognostic assays asdescribed herein. In prophylactic applications, pharmaceuticalcompositions or medicaments of aromatic-cationic peptides areadministered to a subject susceptible to, or otherwise at risk of adisease or condition in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. Administration of a prophylacticaromatic-cationic can occur prior to the manifestation of symptomscharacteristic of the aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending uponthe type of aberrancy, e.g., a aromatic-cationic peptide which acts toenhance or improve mitochondrial function can be used for treating thesubject. The appropriate compound can be determined based on screeningassays described herein.

Therapeutic Methods.

Another aspect of the invention includes methods of modulating insulinresistance or sensitivity in a subject for therapeutic purposes. Intherapeutic applications, compositions or medicaments are administeredto a subject suspected of, or already suffering from such a disease inan amount sufficient to cure, or at least partially arrest, the symptomsof the disease (biochemical, histologic and/or behavioral), includingits complications and intermediate pathological phenotypes indevelopment of the disease. An amount adequate to accomplish therapeuticor prophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with thearomatic-cationic peptide) or, alternatively, in vivo (e.g., byadministering the aromatic-cationic peptide to a subject). As such, theinvention provides methods of treating an individual afflicted with ainsulin resistance-associated disease or disorder.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with a peptide may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vivo methods typically includethe administration of an aromatic-cationic peptide, such as thosedescribed above, to a mammal, preferably a human. When used in vivo fortherapy, the aromatic-cationic peptides of the present invention areadministered to the subject in effective amounts (i.e., amounts thathave desired therapeutic effect). They will normally be administeredparenterally or orally. The dose and dosage regimen will depend upon thedegree of the insulin resistance-related disease or disorder, thecharacteristics of the particular aromatic-cationic peptide used, e.g.,its therapeutic index, the subject, and the subject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide useful in the methods of the presentinvention, preferably in a pharmaceutical composition, may beadministered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds. Thepeptide may be administered systemically or locally.

The aromatic-cationic peptides described herein can be incorporated intopharmaceutical compositions for administration, singly or incombination, to a subject for the treatment or prevention of a disorderdescribed herein. Such compositions typically include the active agentand a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), and transmucosal administration. Solutions or suspensionsused for parenteral, intradermal, or subcutaneous application caninclude the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic. Forconvenience of the patient or treating physician, the dosing formulationcan be provided in a kit containing all necessary equipment (e.g. vialsof drug, vials of diluent, syringes and needles) for a treatment course(e.g. 7 days of treatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The aromatic-cationic peptide compositions can include a carrier, whichcan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thiomerasol, and the like. Glutathione and otherantioxidants can be included to prevent oxidation. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedmy iontophoresis.

A therapeutic protein or peptide can be formulated in a carrier system.The carrier can be a colloidal system. The colloidal system can be aliposome, a phospholipid bilayer vehicle. In one embodiment, thetherapeutic protein is encapsulated in a liposome while maintainingprotein integrity. As one skilled in the art would appreciate, there area variety of methods to prepare liposomes. (See Lichtenberg et al.,Methods Biochem. Anal., 33:337-462 (1988); Anselem et al., LiposomeTechnology, CRC Press (1993)). Liposomal formulations can delayclearance and increase cellular uptake (See Reddy, Ann. Pharmacother.,34 (7-8):915-923 (2000)).

The carrier can also be a polymer, e.g., a biodegradable, bio compatiblepolymer matrix. In one embodiment, the therapeutic protein can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of e.g., collagen, fibronectin, elastin, celluloseacetate, cellulose nitrate, polysaccharide, fibrin, gelatin, andcombinations thereof. In one embodiment, the polymer is poly-lactic acid(PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices canbe prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone(hGH) has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylacetic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods 4 (3) 201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol. 13 (12):527-37 (1995).Mizguchi et al., Cancer Lett. 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Typically, an effective amount of the aromatic-cationic peptides of thepresent invention, sufficient for achieving a therapeutic orprophylactic effect, range from about 0.000001 mg per kilogram bodyweight per day to about 10,000 mg per kilogram body weight per day.Preferably, the dosage ranges are from about 0.0001 mg per kilogram bodyweight per day to about 100 mg per kilogram body weight per day. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight everyday, every two days or every three days or within the range of 1-10mg/kg every week, every two weeks or every three weeks. In oneembodiment, a single dosage of peptide ranges from 0.1-10,000 microgramsper kg body weight. In one embodiment, aromatic-cationic peptideconcentrations in a carrier range from 0.2 to 2000 micrograms perdelivered milliliter. An exemplary treatment regime entailsadministration once per day or once a week. Intervals can also beirregular as indicated by measuring blood levels of glucose or insulinin the subject and adjusting dosage or administration accordingly. Insome methods, dosage is adjusted to achieve a desired fasting glucose orfasting insulin concentration. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the subject shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10⁻¹¹ to 10⁻⁶ molar, e.g., approximately 10⁻⁷molar. This concentration may be delivered by systemic doses of 0.01 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, most preferably by single daily or weekly administration,but also including continuous administration (e.g. parenteral infusionor transdermal application).

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance with the invention can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals, such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In a preferred embodiment, the mammal is ahuman.

Labeled Aromatic-Cationic Peptides and Diagnostic Methods

Disclosed herein are methods comprising providing a labeledaromatic-cationic peptide to a cell or a subject, wherein the peptidehas a detectable label conjugated to a peptide. In one embodiment, aspecific combination of a particular label with a particular peptideallows for detecting localization of the peptide within a cell.

Labeled Aromatic Cationic Peptides.

In one embodiment, the aromatic-cationic peptides of the presentinvention are coupled with a label moiety, i.e., detectable group. Theparticular label or detectable group conjugated to the aromatic-cationicpeptide of the invention is not a critical aspect of the invention, solong as it does not significantly interfere with the specific activityof the aromatic cationic peptide of the present invention. Thedetectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and imaging, in general, most any label usefulin such methods can be applied to the present invention. Thus, a labelis any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texasred, rhodamine, and the like), radiolabels (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I,¹²¹I, ¹¹²In, ^(99m)Tc), other imaging agents such as microbubbles (forultrasound imaging), ¹⁸F, ¹¹C, ¹⁵O, (for Positron emission tomography),^(99m)TC, ¹¹¹In (for Single photon emission tomography), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and calorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, and the like)beads. Patents that described the use of such labels include U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;and 4,366,241, each incorporated herein by reference in their entiretyand for all purposes. See also Handbook of Fluorescent Probes andResearch Chemicals (6^(th) Ed., Molecular Probes, Inc., Eugene Oreg.).

The label can be coupled directly or indirectly to the desired componentof an assay according to methods well known in the art. As indicatedabove, a wide variety of labels can be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, e.g., biotin, thyroxine,and cortisol, it can be used in conjunction with the labeled,naturally-occurring anti-ligands.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds useful as labelingmoieties, include, but are not limited to, e.g., fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, andthe like. Chemiluminescent compounds useful as labelling moieties,include, but are not limited to, e.g., luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal-producing systems which can be used, see, U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels can bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels can be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Diagnostic Applications of Labeled Aromatic Cationic Peptides

In one embodiment, the method comprising administering the labeledaromatic-cationic peptide to a cell or a subject and achieving a desiredlocalization. In one embodiment of the invention, the method comprisingadministering the labeled aromatic-cationic peptide to a human cell andachieving a desired localization. A desired localization refers to alabeled aromatic-cationic peptide being specifically sequestered in adesired cellular component, e.g., the mitochondrion. Those skilled inthe art will recognize that any number of labeled aromatic-cationicpeptides of the present invention may be delivered to a cell and themethod remains within the spirit and scope of the present invention. Inaddition, those skilled in the art will recognize that cellular imagingvarious types of cells from various types of sources are within thespirit and scope of the present invention.

Labeled aromatic-cationic peptides of the invention can be used in vitroand/or in vivo to detect target molecules of interest. In many cases,the labeled aromatic-cationic peptides can simply be added to testsamples in a homogenous assay, not requiring addition of multiplereagents and/or wash steps before detection of the target. Labeledaromatic-cationic peptides of the invention may contact target moleculesor cellular compartments in vitro by simple addition to a test samplecontaining the target molecules or cells. Test samples for in vitroassays can be, e.g., molecular libraries, cell lysates, analyte eluatesfrom chromatographic columns, and the like. The in vitro assay oftentakes place in a chamber, such as, e.g., a well of a multiwell plate, atest tube, an Eppendorf tube, a spectrophotometer cell, conduit of ananalytical system, channels of a microfluidic system, an open array, andthe like.

Where labeled aromatic-cationic peptides of the invention areadministered to living cells, binding can take place with targets on thecell surface or within the cell itself, e.g., the labeledaromatic-cationic peptide is transferred into the cell to make contactwith an intracellular target molecule. In some cases, the labeledaromatic-cationic peptide can penetrate a cell suspected of containing aselected target passively by mere exposure of the cell to a mediumcontaining the labeled aromatic-cationic peptides. In other embodiments,the labeled aromatic-cationic peptide is actively transferred into thecell by mechanisms known in the art, such as, e.g., poration, injection,transduction along with transfer peptides, and the like.

Following contact of the cells with the labeled aromatic-cationicpeptides, the methods may comprise irradiating the cell with an energysource. In one embodiment, the energy source is a light source. In oneembodiment, the imaging agent of the labeled aromatic-cationic peptideis activated by the energy source. In one embodiment, the imaging agentof the labeled aromatic-cationic peptide gives off a detectable signalwhen it is illuminated by the energy source. In one embodiment of theinvention, the imaging agent gives off a detectable fluorescence inresponse to the energy source.

In one embodiment of the invention, the fluorescence given off by theimaging agent in response to the light source may be observed andmeasured. In one embodiment of the invention, the fluorescence isobserved and measured with a confocal microscope. Those skilled in theart will recognize that various devices used to observe and measurefluorescence are within the spirit and scope of the present invention.

EXAMPLES

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1 Mitochondrial Dysfunction in Rats Fed a High Fat Diet

To determine the potential impact of diet-induced obesity on the controlof cellular redox balance in skeletal muscle, a novel approach tomeasure the rate of mitochondrial H₂O₂ emission in permeabilizedskeletal muscle fiber bundles was developed. See Anderson et al., J.Clin Invest (doi:10.1172/JCI37048). During basal (state 4) respirationsupported by NADH-linked complex I substrates, the rate of superoxideformation is low, representing 0.1-0.5% of total O₂ utilization(Anderson & Neufer, Am J Physiol Cell Physiol 290, C844-851 (2006);St-Pierre et al., J Biol Chem 277, 44784-44790 (2002)). However,respiration supported exclusively by succinate, an FADH₂-linked complexII substrate, elicits high rates of superoxide production by generatingreverse electron flow back into complex I (Anderson & Neufer, Am JPhysiol Cell Physiol 290, C844-851 (2006); St-Pierre et al., J Biol Chem277, 44784-44790 (2002); Liu et al., J Neurochem 80, 780-787 (2002);Turrens et al., Biochem J 191, 421-427 (1980)). This Example describesmethods for measuring mitochondrial function in permeabilized muscletissues and examines the effects of a high fat diet on mitochondrialfunction.

Animals and Reagents.

Thirty male Sprague-Dawley rats were obtained from Charles RiverLaboratory (Wilmington, Mass.) and housed in a temperature (22° C.) andlight-controlled room and given free access to food and water. Twenty ofthe original thirty rats were kept on a high (60%) fat diet (ResearchDyets, Bethlehem, Pa.). Skeletal muscle was obtained from anesthetizedanimals (100 mg/kg ip ketamine-xylazine). After surgery, animals werekilled by cervical dislocation while anesthetized. Amplex Red Ultrareagent was obtained from Molecular Probes (Eugene, Oreg.). Stigmatellinand horseradish peroxidase (HRP) were obtained from Fluka Biochemika(Buchs, Switzerland), and all other chemicals were purchased fromSigma-Aldrich (St. Louis, Mo.). All animal studies were approved by theEast Carolina University Institutional Animal Care and Use Committee.

Preparation of Permeabilized Muscle Fiber Bundles.

Briefly, small portions (25 mg) of soleus, RG, and WG muscle weredissected and placed in ice-cold buffer X, containing 60 mM K-MES, 35 mMKCl, 7.23 mM K₂ EGTA, 2.77 mM CaK₂ EGTA, 20 mM imidazole, 0.5 mM DTT, 20mM taurine, 5.7 mM ATP, 15 mM PCr, and 6.56 mM MgCl₂.6H₂O (pH 7.1, 295mosmol/kg H₂O). The muscle was trimmed of connective tissue and cut downto fiber bundles (2×7 mm, 4-8 mg wet wt). With a pair of needle-tippedforceps under a dissecting microscope, fibers were gently separated fromone another to maximize surface area of the fiber bundle, leaving onlysmall regions of contact. To permeabilize the myofibers, each fiberbundle was placed in ice-cold buffer X containing 50 μg/ml saponin andincubated on a rotator for 30 min at 4° C. Following permeabilization,the permeabilized fiber bundles (PmFBs) were washed in ice-cold Buffer Zcontaining (in mM) 110 K-MES, 35 KCl, 1 EGTA, 10 K₂HPO₄, 3 MgCl₂-6H₂O, 5mg/ml BSA, 0.1 glutamate and 0.05 malate (pH 7.4, 295 mOsm) and remainedin Buffer Z on a rotator at 4° C. until analysis (<2 h).

Mitochondrial Respiration and H₂O₂ Emission Measurements.

High resolution respirometric measurements were conducted at 30° C. inBuffer Z using the Oroboros O₂K Oxygraph (Innsbruck, Austria).Mitochondrial H₂O₂ emission was measured at 30° C. during state 4respiration in Buffer Z (10 μg/ml oligomycin) by continuously monitoringoxidation of Amplex Red using a Spex Fluoromax 3 (Jobin Yvon, Ltd.)spectrofluorometer with temperature control and magnetic stirringat >1000 rpm. Amplex Red reagent reacts with H₂O₂ in a 1:1 stoichiometrycatalyzed by HRP to yield the fluorescent compound resorufin and molarequivalent O₂. Resorufin has excitation/emission characteristics of 563nm/587 nm and is extremely stable once formed. After baselinefluorescence (reactants only) was established, the reaction wasinitiated by addition of a permeabilized fiber bundle to 300 μl ofbuffer Z containing 5 μM Amplex Red and 0.5 U/ml HRP, with succinate at37° C. For the succinate experiments, the fiber bundle was washedbriefly in buffer Z without substrate to eliminate residual pyruvate andmalate from the wash. Where stated, 10 μg/ml oligomycin was included inthe reaction buffer to block ATP synthase and ensure state 4respiration. At the conclusion of each experiment, PmFBs were washed inddH₂O to remove salts and freeze-dried in a lyophilizer (LabConco). Therate of respiration is expressed as pmol·s⁻¹·mg dry weight⁻¹, andmitochondrial H₂O₂ emission expressed as pmol·min⁻¹·mg dry weight⁻¹.

Statistical Analyses.

Data are presented as means±SE. Statistical analyses were performedusing a one-way ANOVA with Student-Newman-Keuls method for analysis ofsignificance among groups. The level of significance was set at P<0.05.

Results.

To provide a better measure of the respiratory system's potential togenerate and/or emit H₂O₂ in relation to progressively increasingmetabolic flux (without a change in ATP demand), the changes in H₂O₂emission in response to titration of succinate during state 4respiration supported by the complex I substrates pyruvate and malatewere continuously monitored. By plotting the rate of H₂O₂ emissionversus succinate concentration, it was reasoned that a leftward shift inthe curve would indicate an increase, whereas a rightward shift wouldindicate a decrease, in the oxidant emitting potential of therespiratory system. FIG. 1A shows a representative trace comparing ratesof mitochondrial H₂O₂ emission from permeabilized skeletal muscle fibersprepared from rats fed standard chow, lard 3 days or high fat chow 3weeks. The experiment is started by addition of a small amount ofglutamate and malate (G/M) to a de-energized fiber bundle (FB), andfollowed by successively increasing concentrations (in mM) of succinate.

Surprisingly, switching rats from a standard high carbohydrate chow dietto 100% fat (lard) for 3 days or a 60% high fat diet for 3 weeks induceda remarkable 3 to 4-fold increase in the maximal rate of mitochondrialH₂O₂ emission with little to no change in sensitivity (FIGS. 1A and 1B).Addition of rotenone at the conclusion of succinate titration eliminatedH₂O₂ emission (not shown), confirming complex I as the source ofsuperoxide production from both control and high fat fed rats.Mitochondrial oxidant emitting potential was also measured by titratingpyruvate/malate in the presence of antimycin (complex III inhibitor),again revealing >2-fold higher maximal rate of H₂O₂ emission in high fatfed rats (FIG. 2). These findings demonstrate that the mitochondrialoxidant emitting potential in skeletal muscle is markedly increasedwithin as little as three days after transitioning to a high fat diet.

Example 2 Effects of Aromatic-Cationic Peptides on ROS Production inRats Fed a High Fat Diet

Superoxide production is higher during basal respiration supported byfatty acid versus carbohydrate metabolism, raising the possibility thatthe increase in mitochondrial oxidant emitting potential induced by ahigh fat diet may be precipitated by a persistent elevation in oxidantproduction (i.e., by a ROS-induced ROS release mechanism). To test thishypothesis, the effects of the aromatic-cationic peptide SS-31 onmitochondrial function in high fat fed rats were examined. SS-31 isunique in that it localizes specifically within the mitochondrial innermembrane where it scavenges ROS without affecting membrane potential orrespiratory control. This small peptide antioxidant has been shown toeffectively reduce ROS in hearts subjected to myocardial stunning (Zhaoet al., J Biol Chem 279, 34682-34690 (2004)), in pancreatic islet cellsafter transplantation (Thomas et al., Journal of the American Society ofNephrology 16, TH-FC067 (2005)), and in animal models of Parkinson's andamyotrophic lateral sclerosis disease (Petri et al., J Neurochem 98,1141-1148 (2006); Szeto et al., AAPS J 8, E521-531 (2006)).

Ten rats of the high fat fed group received daily intraperitonealinjections of SS-31 dissolved in phosphate-buffered saline (1.5 mg/kg).Dose response curves for SS-31 were established in vitro (FIG. 3A) andin vivo (FIG. 3B). Mitochondrial function was measured according to themethods described in Example 1. Both dose response curves revealedgreater than 50% reduction in mitochondrial H₂O₂ emission duringsuccinate-supported respiration.

Next, rats were placed on a high fat diet (60%) for six weeks with orwithout daily administration of SS-31. Succinate titration experimentsconducted on permeabilized fibers again revealed a remarkable 3-foldincrease in the maximal rate of H₂O₂ emission in high fat fed rats (FIG.3C). Permeabilized fibers from high fat fed rats also generated nearly a2-fold greater rate of H₂O₂ emission during basal respiration supportedby palmitoyl-carnitine (FIG. 3D). However, in high fat fed rats treatedwith SS-31, the increase in mitochondrial oxidant emitting potentialduring both succinate and palmitoyl-carnitine supported respiration wascompletely prevented (FIGS. 3C & 3D). Basal respiration supported bypyruvate/malate was slightly increased in fibers from high fat fed rats,suggesting some degree of uncoupling (FIG. 3E). However, in high fat fedrats, basal rates of pyruvate/malate- or palmitoyl-carnitine-supportedrespiration were not affected by SS-31 treatment (FIGS. 3E and 3F),indicating that the normalization of H₂O₂ emission with SS-31 treatmentwas not mediated by an increase in proton leak. SS-31 treatment also didnot affect the weight gain in high fat fed rats (data not shown).Collectively, these findings demonstrate that administration of amitochondrial targeted antioxidant, such as the aromatic-cationicpeptides of the invention, is sufficient to prevent or compensate forthe increase in mitochondrial oxidant emitting potential induced by ahigh fat diet. As such, administration of the aromatic-cationic peptidesof the present invention is useful in methods of preventing or treatinginsulin resistance caused by mitochondrial dysfunction in mammaliansubjects.

It is increasingly recognized that the intracellular localization andactivity of many proteins (e.g., receptors, kinases/phosphatases,transcription factors, etc.) is reversibly controlled by the oxidationstate of specific thiol (—SH)-containing residues, leading to theconcept that shifts in the intracellular redox environment affect theoverall biological status of the cell (Schafer and Buetner, Free RadicBiol Med 30, 1191-1212 (2001)). Glutathione (GSH), the most abundantredox buffer in cells, is reversibly oxidized to GSSG by glutathioneperoxidase in the presence of H₂O₂, and reduced back to GSH byglutathione reductase with electrons donated by NADPH. The ratio ofGSH/GSSG is very dynamic, largely reflecting the overall redoxenvironment of the cell.

Protein homogenates were prepared by homogenizing 100 mg of poweredfrozen muscle in a buffer containing in mM: 10 Tris, 1 EDTA, 1 EGTA, 2NaOrthovanadate, 2 NaPyrophosphate, 5 NaF, protease inhibitor cocktail(Complete) at pH 7.2. After homogenization, 1% Triton X-100 was added tothe protein suspension, cotexed and allowed to sit on ice for 5 minutes.The tubes were then spun at 10,000 rpm for 10 minutes to pellet theinsoluble debris. For GSSG measurement, tissue was homogenized in asolution containing 20 mM Methyl-2-vinylpyridinium triflate to scavengeall reduced thiols in the sample. Total GSH and GSSG were then measuredusing the reagents and calibration set provided by the GSH/GSSG assay(Oxis Research) according to the manufacturer's instructions, with smallmodifications as needed.

Surprisingly, it was found that high fat feeding resulted in an ˜30%reduction in total cellular glutathione content (GSH_(t)) irrespectiveof SS-31 treatment (FIG. 3G), demonstrating that high fat intakecompromises GSH-mediated redox buffering capacity in skeletal muscle. Toestablish a link between the increased mitochondrial oxidant emissionbrought about by high fat diet and its effect on overall redoxenvironment of skeletal muscle, both GSH and GSSG were measured inskeletal muscle of chow fed and high fat fed rats under two conditions;after a 10 h fast and 1 h after administration of a standard glucoseload (oral gavage, 10 h fasted). In chow fed controls, glucose ingestionelicited an ˜50% reduction in the GSH/GSSG ratio (normalized to GSH_(t),FIG. 3H), presumably reflecting an acute shift to a more oxidized statein response to the increase in insulin-stimulated glucose metabolism. Inhigh fat fed rats, the GSH/GSSG ratio was already reduced by ˜50% in the10 h fasted state relative to chow fed controls and declined further inresponse to the glucose ingestion. SS-31 treatment, however, preservedthe GSH/GSSG ratio near control levels, even in response to glucoseingestion. These findings demonstrate that a high fat diet shifts theintracellular redox environment in skeletal muscle to a more oxidizedstate. Treatment with SS-31 was able to preserve the intracellular redoxstate in skeletal muscle, presumably by scavenging primary oxidants andthereby compensating for the reduction in total GSH-mediated redoxbuffering capacity induced by a high fat diet. Thus, administration of amitochondrial-targeted antioxidant, such as the aromatic-cationicpeptides of the invention, either prevents or compensates for themetabolic dysfunction that develops in rats fed a high fat diet. Assuch, administration of the aromatic-cationic peptides of the presentinvention is useful in methods of preventing or treating this metabolicdysfunction in mammalian subjects.

Example 3 Oral Glucose Tolerance Tests

To determine whether mitochondrial-derived changes in intracellularredox environment may be linked to the etiology of high fat diet-inducedinsulin resistance, oral glucose tolerance tests were performed in ratsafter the six week high fat diet. On the day of experiments, food wasremoved 10 hr prior to administration of a 2 g/kg glucose solution viagavage. Glucose levels were determined on whole blood samples (Lifescan,Milpitas, Calif.). Serum insulin levels were determined via a rat/mouseELISA kit (Linco Research, St. Charles, Mo.). Fasting data were used todetermine homeostatic model assessment (HOMA)—calculated as fastinginsulin (μU/ml)× fasting glucose (mM)/22.5.

Blood glucose (FIG. 4A) and insulin (FIG. 4B) responses to the oralglucose challenge were higher and more sustained in high fat fed ratscompared with standard chow-fed rats. Treatment of high fat fed ratswith SS-31 normalized both the blood glucose and insulin responses tothe oral glucose challenge.

Increased homeostatic model assessment (HOMA, FIG. 4C), and greater areaunder the curves for both blood glucose and insulin (FIG. 4D) confirmedthe development of insulin resistance in high fat fed rats. Treatment ofhigh fat fed rats with SS-31 completely blocked the development ofinsulin resistance (FIGS. 4C and 4D). To further assess insulinsensitivity, the phosphorylation state of the insulin signaling proteinAkt in skeletal muscle from animals was measured after a 10 h fast or 1h after receiving an oral glucose load. In response to glucoseingestion, Akt phosphorylation increased ˜5-fold in skeletal muscle ofchow-fed controls but was unchanged in high fat fed rats (FIGS. 4E and4F), confirming the presence of insulin resistance at the level ofinsulin signaling. Treatment of high fat fed rats with SS-31 completelypreserved Akt phosphorylation in response to glucose ingestion (FIGS. 4Eand 4F), again indicating preservation of insulin sensitivity. Thus,administration of a mitochondrial-targeted antioxidant, such as thearomatic-cationic peptides of the invention, prevents insulin resistancethat develops in rats fed a high fat diet. As such, administration ofthe aromatic-cationic peptides of the present invention is useful inmethods of preventing or treating insulin resistance in mammaliansubjects.

Example 4 Mitochondrial Dysfunction in Human Subjects

To strengthen the link between mitochondrial-derived changes inintracellular redox environment and insulin resistance, and to see ifthe same phenomena is translatable to humans, the control ofmitochondrial H₂O₂ emission and respiration in permeabilized skeletalmyofiber bundles obtained by muscle biopsy from lean, insulin sensitive(BMI=21.6±1.2 kg·m⁻², HOMA=1.2±0.4) and obese, insulin resistant(BMI=43.0±4.1 kg·m⁻², HOMA=2.5±0.7, P<0.05) male human subjects wasmeasured.

Eight healthy men (ages 18-31 y) of mixed race were recruited toparticipate in this investigation: five were classified as lean(BMI≦24.9 kg/m²) and three were classified as morbidly obese (BMI≧35kg/m²). All participants were non-smokers with no history of metabolicdisease. None of the subjects had any diseases or were taking anymedications known to alter metabolism. On the day of the experiment,subjects reported to the laboratory following an overnight fast(approximately 12 h). A fasting blood sample was obtained fordetermination glucose and insulin (Labcorps). Height and body weight wasrecorded and skeletal muscle biopsies were obtained from lateral aspectof vastus lateralis by the percutaneous needle biopsy technique underlocal subcutaneous anesthesia (1% lidocaine). A portion of the biopsysamples were flash frozen in liquid N₂ for protein analysis, and anotherportion used to prepare permeabilized fiber bundles.

Results.

Mitochondrial H₂O₂ emission was ˜2-fold higher in obese versus lean inresponse to titration of succinate (FIG. 5A) and nearly 4-fold higherduring basal respiration supported by fatty acid (FIG. 5B). Despite thedifference in H₂O₂ emission, basal O₂ utilization was similar betweenlean and obese subjects (FIG. 5C); consequently, the rate ofmitochondrial free radical leak was ˜2-fold higher duringglutamate/malate/succinate and >4-fold during palmitoyl-carnitinesupported basal respiration (FIG. 5D). Maximal ADP-stimulated O₂consumption was ˜35% lower in permeabilized myofibers from the obesesubjects during respiration supported by the complex I substratesglutamate/malate (FIG. 5C), consistent with the overall reduced skeletalmuscle respiratory capacity associated with obesity. Finally, similar torats fed a high fat diet, both total cellular GSH content and theGSH/GSSG ratio were ˜50% lower in skeletal muscle of obese humans (FIGS.5E and 5F), indicative of both an overall lower redox buffer capacityand a decidedly more oxidized intracellular redox environment.

In summary, these findings collectively establish mitochondrial ROSemission and the resulting shift to a more oxidized skeletal muscleredox environment as an underlying cause of high fat diet-inducedinsulin resistance. An increase in the H₂O₂ emitting potential ofmitochondria appears to be a primary factor contributing to this shiftin redox environment. Thus, administration of a mitochondrial-targetedantioxidant such as the aromatic-cationic peptides of the invention,either prevents or compensates for the metabolic dysfunction thatdevelops with over nutrition. As such, administration of thearomatic-cationic peptides of the present invention is useful in methodsof preventing or treating insulin resistance in human subjects.

Example 5 Prevention and Treatment of Insulin Resistance byAromatic-Cationic Peptides of the Invention in the Zucker Rat Model

To further demonstrate the prevention of insulin resistance on the onehand, and treatment of insulin resistance on the other hand, thearomatic-cationic peptides of the invention are tested on the fatty(fa/fa) Zucker rat, a model of diet-induced insulin resistance. Comparedwith the 6 wk high fat fed Sprague-Dawley rat model (as used in Examples1-3), the fatty Zucker rats develop a much greater degree of obesity andinsulin resistance. As in the high fat fed rats, mitochondrialdysfunction (increased oxidant emitting potential) is also evident inpermeabilized fibers from fatty Zucker rats.

First, to examine the effects of the aromatic-cationic peptides of theinvention on prevention of insulin resistance, young Zucker rats (˜3-4weeks of age) are administered SS-31 for approximately 6 weeks. As theseyoung rats do not yet exhibit signs or symptoms of insulin resistance,they provide a useful model for assessing the efficacy of methods ofpreventing insulin resistance. SS-31 (1.0-5.0 mg/kg body wt) isadministered to the rats via i.p. or oral administration (i.e., drinkingwater or gavage).

It is predicted that SS-31 administration will attenuate or prevent thedevelopment of whole body and muscle insulin resistance that normallydevelops in the fatty (fa/fa) Zucker rat. Measured outcomes include bodyweight, fasting glucose/insulin/free fatty acid, glucose tolerance(OGTT), in vitro muscle insulin sensitivity (incubation), markers ofinsulin signaling (Akt-P, IRS-P), mitochondrial function studies onpermeabilized fibers (respiration, H₂O₂ emission), markers ofintracellular oxidative stress (lipid peroxidation, GSH/GSSG ratio,aconitase activity) and mitochondrial enzyme activity. A comparison ismade between control rats and fa/fa rats administered SS-31. Controlsinclude wild-type and fa/fa rats not administered SS-31. Successfulprevention of insulin resistance by the aromatic-cationic peptides ofthe invention is indicated by a reduction in one or more of the markersassociated with insulin resistance or mitochondrial dysfunctionenumerated above.

Second, to examine the effects of the aromatic-cationic peptides of theinvention on treatment of insulin resistance, Zucker rats (˜12 weeks ofage) are administered SS-31 for approximately 6 weeks. As these ratsshow signs of obesity and insulin resistance, they provide a usefulmodel for assessing the efficacy of methods of treating insulinresistance. SS-31 (1.0-5.0 mg/kg body wt) is administered to the ratsvia i.p. or oral administration (i.e., drinking water or gavage).

It is predicted that SS-31 administration will ameliorate or reducewhole body and muscle insulin resistance that normally develops in theseolder fatty (fa/fa) Zucker rats. Measured outcomes include body weight,fasting glucose/insulin/free fatty acid, glucose tolerance (OGTT), invitro muscle insulin sensitivity (incubation), markers of insulinsignaling (Akt-P, IRS-P), mitochondrial function studies onpermeabilized fibers (respiration, H₂O₂ emission), markers ofintracellular oxidative stress (lipid peroxidation, GSH/GSSG ratio,aconitase activity) and mitochondrial enzyme activity. A comparison ismade between control rats and fa/fa rats administered SS-31. Controlsinclude wild-type and fa/fa rats not administered SS-31. Successfultreatment of insulin resistance by the aromatic-cationic peptides of theinvention is indicated by a reduction in one or more of the markersassociated with insulin resistance or mitochondrial dysfunctionenumerated above.

EQUIVALENTS

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method of treating or preventing insulinresistance in a mammalian subject, comprising administering to saidmammalian subject a therapeutically effective amount of the peptideD-Arg-2′6′Dmt-Lys-Phe-NH₂ (SS-31).
 2. The method of claim 1, wherein thesubject is a human.
 3. The method of claim 1, wherein the subject issuffering from diet-induced insulin resistance.
 4. The method of claim1, wherein the insulin resistance is associated with type II diabetes.5. The method of claim 1, wherein the insulin resistance is associatedwith obesity.
 6. The method of claim 1, wherein the insulin resistanceis associated with coronary artery disease, renal dysfunction,atherosclerosis, hyperlipidemia, essential hypertension, or fatty liver.7. The method of claim 1, wherein the insulin resistance is drug-inducedinsulin resistance.
 8. The method of claim 1, wherein the peptide isadministered prior to the onset of type II diabetes.
 9. The method ofclaim 1, wherein the peptide is administered orally, topically,systemically, intravenously, subcutaneously, or intramuscularly.
 10. Themethod of claim 1 further comprising the step of identifying the mammalin need of treating or preventing insulin resistance.
 11. The method ofclaim 1 further comprising the step of monitoring the mammal fortreatment or prevention of insulin resistance.
 12. The method of claim1, wherein the therapeutically effective amount provides a concentrationof peptide in a target tissue of about 10⁻⁸ to 10⁻⁶ molar.